PIVKA-II refers to a protein classified as prothrombin, which is the blood coagulation factor II, without coagulation factor activity, and is also referred to as abnormal prothrombin. Prothrombin is synthesized in liver and the generation process requires conversion of glutamic acid (Glu) residues into γ-carboxyglutamic acid (Gla) residues by vitamin K-dependent γ-glutamyl carboxylase. Although 10 Gla residues are present near the N-terminus of normal prothrombin, PIVKA-II has all or a portion of 10 residues not converted into Gla and remaining as Glu residues. PIVKA-II was initially found in the blood of vitamin K deficient or vitamin K antagonist-treated patients. Since the blood concentration increases in association with hepatoma, PIVKA-II is recently measured as a tumor marker of hepatoma. PIVKA-II is an abbreviation of protein induced by Vitamin K absence or antagonists-II and is also referred to as des-γ-carboxy prothrombin (DCP) (References: Weitz, I. C., and Liebman, H. A., (1993) Hepatology 18, 990-997; Suzuki M, Shiraha H, Fujikawa T, Takaoka N, Ueda N, Nakanishi Y, Koike K, Takaki A, Shiratori Y, J Biol Chem, 2005 Feb. 25; 280 (8), 6409-15; A. Nakao, A. Virji, Y. Iwaki, B. Carr, S. Iwatsuki, and E. Starzl, Hepatogastroenterology, 1991 October, 38(5), 450-453).
As methods for specifically measuring PIVKA-II in a sample, presently known are a method of separating prothrombin and PIVKA-II by using HPLC (Anal Biochem, 1984 February; 137(1), 227-9), a method of separating prothrombin and PIVKA-II with polyacrylamide gel-affinity electrophoresis using calcium lactate (Non-Patent Literature 1), an ELISA-based method using an antibody specifically reactive with PIVKA-II (DCP) (Non-Patent Literature 2), etc.
In the method of Non-Patent Literature 1, calcium lactate is used at the time of electrophoresis and this is intended to generate a difference in electrophoretic mobility between normal prothrombin and PIVKA-II due to the presence of calcium-binding capacity of the Gla residues so as to separate the both.
In Non-Patent Literature 2, PIVKA-II is measured by using an antibody (C4B6) specifically binding to PIVKA-II only under the presence of calcium ions. However, only the C4B6 antibody is reported as an anti-PIVKA-II antibody requiring the presence of calcium ions for specifically measuring PIVKA-II until now. Therefore, the addition of calcium is usually not required for using an antibody specifically reactive with PIVKA-II (DCP) in a measuring method of PIVKA-II.
The current mainstream of methods for specifically measuring PIVKA-II in a sample is a method based on EIA (enzyme immunoassay), RIA (radioimmunoassay), or ELISA (enzyme-linked immunoassay) of measuring through a two-step sandwich method using a monoclonal antibody specifically reactive with PIVKA-II (DCP) and an anti-prothrombin polyclonal antibody (e.g., JP H05-43357 A (Translation of PCT Application) and JP H09-43237 A).
Calcium is not used in the measuring methods of JP H05-43357 A (Translation of PCT Application), JP H09-43237 A, and International Publication Pamphlet No. WO 2010/104815 based on ELISA and it is generally considered that calcium ions are unnecessary for measurement of PIVKA-II.
Patent Literature 1 is an example of measuring PIVKA-II by utilizing agglutination of carrier particles. A summary of the technique of Patent Literature 1 is as follows. First, a sample is added to magnetic particles carrying a PIVKA-II specific antibody to bind PIVKA-II in the sample to the antibody. At this point, normal prothrombin in the sample is not bound to the magnetic particles. The magnetic particles are trapped by a magnet and washed to remove normal prothrombin. Fluorescent-labeled particles carrying an anti-prothrombin antibody reactive with both PIVKA-II and normal prothrombin is then added to the magnetic particles. As a result, a sandwich structure is formed with the magnetic particles and the fluorescent-labeled particles bound through the two antibodies to PIVKA-II. The sandwich structure is trapped by a magnet and washed to remove the free fluorescent-labeled particles. Lastly, the sandwich structure is alkali-treated to dissociate the fluorescent-labeled particles from PIVKA-II to measure fluorescence intensity. The fluorescence intensity is proportional to PIVKA-II concentration in the sample. As described above, the method described in Patent Literature 1 is a heterogeneous measuring method having the steps of B/F separation/washing and employs as the detection principle the measurement of fluorescence intensity derived from the fluorescent-labeled particles rather than utilizing as the detection principle the optical measurement of agglutination of the carrier particles itself.
Patent Literature 2 teaches in Examples a basic technique of latex agglutination test employing as the detection principle the optical measurement of agglutination of carrier particles itself in a homogeneous system without the need of the steps of B/F separation/washing. The document teaches “a highly-sensitive immunoassay method characterized in that two different types of monoclonal antibodies to human CEA are carried by two types of latex carriers and are reacted with human CEA in a water solvent to selectively agglutinate the conjugates of the latex carriers and human CEA, wherein the two types of latex particles have average particle diameters different from each other and within a range of 0.05 to 0.500 μm and said monoclonal antibodies are carried by the respective latex particles”.